Oxidative stress and lipid peroxidation occur as a result of ethanol metabolism and have been linked to many diseases. Aldehyde dehydrogenases (ALDHs) are enzymes that play major roles in detoxifying reactive aldehydes produced during these processes. ALDH3B1 belongs to the ALDH family, however, its properties and funcitonal significance remain unknown. The long term goal is to comprehensively characterize ALDH3B1 and investigate its role in oxidative stress. The specific hypothesis is that ALDH3B1 has a functional role in the liver and represents an important cellular defense system against oxidative stress and reactive aldehydes. This hypothesis is based on the preliminary observations that 1) ALDH3B1 is highly expressed in the liver, 2) ALDH3B1 is catalytically active with lipid peroxidaiton-derived aldehydes and 3) ALDH3B1 protects cells from cytoxicity induced by these aldehydes. Specific Aims: 1. Purify and biochemically characterize recombinant human ALDH3B1. ALDH3B1 will be purified from baculovirus-infected Sf9 cells using affinity chromatography. Enzymatic activity, inhibitor-sensitivity and cofactor preference will be evaluated spectrophotometrically in the presence of various aldehyde substrates. 2: Determine the tissue- and cell-specific expression and subcellular localization of ALDH3B1 in C57BU6J mice. The tissue expression of ALDH3B1 protein and mRNA will be analyzed by Western blot and RT-PCR techniques, respectively. Cell-specific expression of ALDH3B1 will be assessed in organ sections by immunohistochemistry. ALDH3B1 subcellular localization will be determined by subcellular fractionation of organs followed by Western blot and immunohistochemistry followed by confocal microscopy. 3: Evaluate the protective role of ALDH3B1 in oxidative stress in vitro. Stable ALDH3B1- and mocktransfected Hepa-1 and HEK293 cell lines will be treated with various aldehydes and cytotoxicity, apoptosis, protein oxidation and lipid peroxidation will be measured using cellular assays. The use of siRNA in cells expressing endogenous ALDH3B1 will be utilized to assess the protective role of ALDH3B1. The regulation of ALDH3B1 by ethanol will be examined in cells expressing endogenous ALDH3B1. Cells will be treated with ethanol and ALDH3B1 protein and mRNA expression will be analyzed by Western blot and RT-PCR. Relevance to Public Health: Alcohol consumption is implicated in the etiology of many human diseases and ethanol metabolism results in the formation of many toxic products. Aldehyde dehydrogenase (ALDH) enzymes play a major role in cell survival by detoxifying these products. The properties of ALDH3B1 and its functional role in these processes will be investigated. [unreadable] [unreadable] [unreadable]